3 Methods
3.1 Running the Test
Reaction
1. The instrument should be programmed to execute 15–20
injections, including a small pre-injection (see Note 7), in
volumes that use fully the 40 μL capacity of the syringe. In
this way, the final concentration of tri-acetyl glucosamine in the
iTC200 will approach ~130 μM representing a considerable
molar excess over the lysozyme. Injections are spaced by 180 s.
The melting temperature of lysozyme at pH 5 in this buffer is
~75
C and so the test can be performed over a wide range of
temperatures.
2. The differential power level to the sample cell should be set to
the middle of the instrument’s measurement range, which for
the iTC200 is 6 μcal/s (see Note 11). The ability to control this
level is important for experiments generating large heats, but
for biological systems at typical concentrations the signals are
typically more modest and so this setting will reliably capture
exothermic or endothermic interactions. There is also an
option to control the data density (time interval for averaging
differential power into a point) and the instrumental “feedback
gain” that sets levels of electrical filtering on the measured
signal. It is best practice to set intervals of 1–2 s and feedback
gain at its fastest level, both producing more “noisy” data. This
allows use of the data in kinetic analysis, either for simple visual
inspection or the more detailed analysis discussed below.
Indeed it is true of any time-based measurement that inappropriate choice of data interval and excessive electrical damping
(slow instrument response) during collection will mask kinetic
information that cannot then be recovered. In contrast, the
cosmetic effects of reducing noise can easily be obtained post
data collection with the use of software.
3. Once the experiment is running, the differential power level is a
good indication of how successful the loading has been. The
various power levels of the instrument, including that of the
final feedback heaters, are configured during instrument construction with both cells containing aqueous solutions and are
therefore a measure of the heat capacity of the sample cell. If
the background baseline differential power levels are lower
than configured in the experiment (>1 μcal/s off) then this
suggests that the sample cell may contain one or more micro
bubbles; the power to this cell is reduced because of the very
low heat capacity of air compared to aqueous solution. Equally,
if the power levels are higher than expected then this can be
indicative of poor loading of the reference cell.
Isothermal Titration Calorimetry
141
3.1 Running the Test
Reaction
1. The instrument should be programmed to execute 15–20
injections, including a small pre-injection (see Note 7), in
volumes that use fully the 40 μL capacity of the syringe. In
this way, the final concentration of tri-acetyl glucosamine in the
iTC200 will approach ~130 μM representing a considerable
molar excess over the lysozyme. Injections are spaced by 180 s.
The melting temperature of lysozyme at pH 5 in this buffer is
~75
C and so the test can be performed over a wide range of
temperatures.
2. The differential power level to the sample cell should be set to
the middle of the instrument’s measurement range, which for
the iTC200 is 6 μcal/s (see Note 11). The ability to control this
level is important for experiments generating large heats, but
for biological systems at typical concentrations the signals are
typically more modest and so this setting will reliably capture
exothermic or endothermic interactions. There is also an
option to control the data density (time interval for averaging
differential power into a point) and the instrumental “feedback
gain” that sets levels of electrical filtering on the measured
signal. It is best practice to set intervals of 1–2 s and feedback
gain at its fastest level, both producing more “noisy” data. This
allows use of the data in kinetic analysis, either for simple visual
inspection or the more detailed analysis discussed below.
Indeed it is true of any time-based measurement that inappropriate choice of data interval and excessive electrical damping
(slow instrument response) during collection will mask kinetic
information that cannot then be recovered. In contrast, the
cosmetic effects of reducing noise can easily be obtained post
data collection with the use of software.
3. Once the experiment is running, the differential power level is a
good indication of how successful the loading has been. The
various power levels of the instrument, including that of the
final feedback heaters, are configured during instrument construction with both cells containing aqueous solutions and are
therefore a measure of the heat capacity of the sample cell. If
the background baseline differential power levels are lower
than configured in the experiment (>1 μcal/s off) then this
suggests that the sample cell may contain one or more micro
bubbles; the power to this cell is reduced because of the very
low heat capacity of air compared to aqueous solution. Equally,
if the power levels are higher than expected then this can be
indicative of poor loading of the reference cell.
Isothermal Titration Calorimetry
141